Intracellular calcium ([Ca2+]i), ph ([pH]i), and cAMP are key regulators of sperm motility. We have documented that sperm cAMP levels increase, [CVa2+]i dramatically declines, and [pH]i increases during epididymal maturation. An increase in sperm [pH]i, while lowering [Ca2=]i, is also a permissive event that enables the elevation of cAMP levels to be translated into motility in caput epididymal sperm in vitro. We suggest that the critical changes in [Ca2+]i during epididymal transit, in turn, increase cAMP levels by calmodulin-mediated mechanisms and in addition amplify the effect of cAMP at the level of protein phosphorylation. Our results clearly indicate the [Ca2+]i, [pH]i, and cAMP interact in sperm to promote epididymal development of sperm motility. We will determine the mechanistic basis for and the functional consequences of the interrelationships among these key regulators during sperm maturation in the bovine epididymis. In aim one, we will quantify the motility response to precise alterations in the intra-sperm levels, either individually or in selected combination, of cAMP, Ca2+, and pH. The flash-photolysis equipment needed to effect alterations in the levels of these regulators has been assembled and the feasibility of this novel technique in sperm has now been documented. A fully automated computerized system, also developed in this laboratory, will be used for analysis of sperm head and flagellar motion. The purpose of this aim is to identify the necessary and sufficient conditions for motility induciton in immotile caput apididymal sperm. In aim two, we will determine how [pH]i and [Ca2+]i are regulated in both mature and immature sperm and are altered during epididymal sperm maturation. Our knowledge of how the levels of these mediators are regulated in mammalian sperm is surprisingly poor. We will determine how [pH]i induced changes in mitochondrial calcium cycling and Na+/Ca2+ antiport mechanisms lower [Ca2+]i during sperm maturation. Finally, we will document how these ions are involved in regulation of intrasperm levels of cAMP. In the final aim, we will extend our novel observations ont he role of okadaic acid-sensitive protein phosphatases in regulating mature sperm motility and epididymal motility development. Protein phosphatases are important mediators in the cross-talk between intracellular second messengers in somatic cells. We will use a combination of biochemical and molecular approaches to identify the phosphatases important to sperm development. Successful execution of these aims will provide the first description of the biochemical basis for the development of sperm motility in the epididymis of any species.